Transplantation of cells into a spinal cord injury (SCI) is a promising approach to promote axonal regeneration across the injury in which continuity of fiber tracts has been interrupted. How effective this transplantation will be will depend upon the interfaces that form between the implant and the host tissue. If these interfaces are not permissive for the regenerating axons, then the cell therapy will not be suitably efficacious. Schwann cells (SCs) are clinically relevant because they can be transplanted into humans autologously; in rats they reduce large cysts that form following SCI, protect tissue from secondary damage, promote axonal regeneration across the injury, provide myelin and modestly improve hindlimb movements (Bunge 2008, Fortun et al 2009a). Our rat work so far has demonstrated that descending brainstem axons are able to cross the rostral host/SC interface and extend along the SC bridge following combination treatments or by simply injecting the SCs in fluid matrigel into the complete transection gap without additional interventions (as described in the Progress Report). The extension of GFAP+ processes into the SC bridge is key to creating a permissive rostral interface for brainstem axons to cross. But these regenerated axons do not cross the caudal interface. Regenerative failure is likely due to the intrinsic failure of adult axons to grow across the impenetrable host/SC caudal interface. The goals of this proposal are to: 1) modify the caudal interface to enable brainstem axons to cross it and grow into the spinal cord; and 2) enhance the intrinsic growth properties of injured adult brainstem neurons. To achieve these goals we will test three different strategies to make the caudal interface more permissive to axon growth and clinically relevant deep brain stimulation (DBS) and genetic approaches to enhance the intrinsic growth potential of injured brainstem neurons. First, SCs from adult nerve will be stimulated with a culture medium (developed in the Monje laboratory) that causes them to revert to an earlier precursor stage and then implanted. Second, inhibiting NF-kB and Eph receptor expression in SCs will be tested as a way to modify one side of the caudal interface. Third, the other side of this interface will be specifically modified by reducing NF-kB expression in astrocytes genetically or pharmacologically. Finally, we will combine the most effective strategy to enhance intrinsic growth of injured brainstem axons with the best strategy to modify the caudal interface to seek the most effective therapies. Outcome measures include assessment of SC and astrocyte co-mingling at the interfaces, counting GFAP+ processes that extend into the SC bridge, and counting GFP+ brainstem axons that regenerate across the interfaces. When axons are found to exit the SC bridge, retrograde tracing to identify the responding parent neurons and electrophysiological, locomotor (BBB, catwalk), sensory (mechanical and thermal) and autonomic (blood pressure, heart rate in response to colon distension) testing will be performed. Discovery of translational methods to improve exiting of regenerated brainstem axons from the SC bridge into the cord will have an important impact on fostering spinal cord repair, particularly for the use of SCs to mend human SCIs. PUBLIC HEALTH RELEVANCE: Cell transplantation is a promising approach to promote new axon growth across the SCI, thereby improving functional outcome. SCs are clinically relevant because they can be transplanted autologously to avoid immune rejection; in rat cords they reduce large cysts that form, protect tissue from further damage, promote axonal regeneration across the injury, provide myelin and modestly improve walking. A bridge of SCs inserted into a complete transection of the rat spinal cord enables brainstem axon regeneration from the cord into the bridge. We have found that the extension of astrocyte processes into the bridge helps brainstem axons to regenerate into the bridge. But these axons do not leave the SC-bridge to grow back into the spinal cord. The goals of this proposal are to modify the interface between the caudal portion of the bridge and the spinal cord and also to enhance the intrinsic growth of axons to enable the regenerated axons to leave the bridge and grow into the caudal spinal cord. Two independent approaches will be used to enhance the intrinsic growth potential of axons: 1) clinically relevant DBS and 2) modification of pathways known to inhibit axon growth (e.g., PTEN). These strategies will be used in combination with transplanted SCs that have been treated to revert to a more immature state and modify signaling inside SCs and astrocytes at their interface. Quantitative histological measures will be employed and, when growth of axons is found beyond the caudal interface, will be combined with retrograde tracing and electrophysiological, locomotor, sensory and autonomic function testing. To find new ways to improve exiting of regenerated axons from the SC-bridge into the cord will have a significant impact on overcoming functional deficits caused by SCI.